Dataset Information

Oviductal vs. ovarian epithelial transformation yields very different tumor phenotypes in Apc fl/fl;Pten fl/fl mice

ABSTRACT: We treated 6-8 week old mice that had floxed alleles of both Apc and Pten (for both alleles in each case) that also carry an Ovgp1-iCre-ERT2 transgene, with one of two treatments; a third group received neither treatment. The Ovgp1-iCre-ERT2 expresses Cre recombinase fused to a tamoxifen-inducible fragment of the estrogen receptor, in tissues where the Ovgp1 gene (oviductal glycoprotein 1) is expressed, which is almost exclusively in mouse oviductal epithelium (equivalent to human fallopian tube epithelium = FTE). Treating the mice with tamoxifen permits the Cre recombinase to enter the cell nucleus and inactivate the Apc and Pten genes. Six of the mice were treated with intraperitoneal injection of tamoxifen (0.1g/kg of body weight) dissolved in corn oil on days 1 and 3 and developed oviductal tumors (OdT) yielding 6 of the samples. Four mice (yielding 5 samples) were instead injected with 50 million plaque-forming units of replication-incompetent AdCre into both ovarian bursal cavities on day 1, which inactivated Apc and Pten in the ovarian surface epithelium (OSE), and lead to ovarian tumors (OT). Ovaries were also harvested from four untreated 6-8 week old mice with the same genotype, with two ovaries from each mouse comprising one control sample. RNA was purified from tumor or normal tissue, and targets for Affymetrix arrays synthesized from the mRNAs. We used Affymetrix Mouse Gene 2.1 ST arrays, which hold 41345 probe-sets, but we largely analyzed just those 25216 probe-sets that were mapped to Entrez gene IDs. Raw data was processed with Robust Multi-array Average algorithm (RMA). Data is log2-transformed transcript abundance estimates. We fit a one-way ANOVA model to the three groups of samples. We supply a supplementary excel workbook that holds the same data as the data matrix file, but also holds the probe-set annotation at the time we analyzed the data, and some simple statistical calculations, which select subsets of the probe-sets as differentially expressed. Consumers should consider obtaining more up-to-date probe-set annotation for the array platform. We also provide supplementary excel files that show our simple analysis of GSE6008, which consists of 99 human ovarian tumor samples of 4 types, and 4 normal ovary samples, where we fit an ANOVA model to the 5 groups. In yet another supplementary file we show the correlation between each human tumor and mouse tumor, where we correlate the difference in log2-transformed values of each tumor from the average of the normals for the same species, for just those genes that were 1-to-1 best homologs according to build 68 of NCBI's Homologene, in order to see how much the human tumors resemble the mouse tumors.

ORGANISM(S): Mus musculus

PROVIDER: GSE80085 | GEO | 2016/08/25

SECONDARY ACCESSION(S): PRJNA317778

REPOSITORIES: GEO

ACCESS DATA

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Similar Datasets

Project description:We treated 6-8 week old mice that had floxed alleles of both Apc and Pten (for both alleles in each case) that also carry an Ovgp1-iCre-ERT2 transgene, with one of two treatments; a third group received neither treatment. The Ovgp1-iCre-ERT2 expresses Cre recombinase fused to a tamoxifen-inducible fragment of the estrogen receptor, in tissues where the Ovgp1 gene (oviductal glycoprotein 1) is expressed, which is almost exclusively in mouse oviductal epithelium (equivalent to human fallopian tube epithelium = FTE). Treating the mice with tamoxifen permits the Cre recombinase to enter the cell nucleus and inactivate the Apc and Pten genes. Six of the mice were treated with intraperitoneal injection of tamoxifen (0.1g/kg of body weight) dissolved in corn oil on days 1 and 3 and developed oviductal tumors (OdT) yielding 6 of the samples. Four mice (yielding 5 samples) were instead injected with 50 million plaque-forming units of replication-incompetent AdCre into both ovarian bursal cavities on day 1, which inactivated Apc and Pten in the ovarian surface epithelium (OSE), and lead to ovarian tumors (OT). Ovaries were also harvested from four untreated 6-8 week old mice with the same genotype, with two ovaries from each mouse comprising one control sample. RNA was purified from tumor or normal tissue, and targets for Affymetrix arrays synthesized from the mRNAs. We used Affymetrix Mouse Gene 2.1 ST arrays, which hold 41345 probe-sets, but we largely analyzed just those 25216 probe-sets that were mapped to Entrez gene IDs. Raw data was processed with Robust Multi-array Average algorithm (RMA). Data is log2-transformed transcript abundance estimates. We fit a one-way ANOVA model to the three groups of samples. We supply a supplementary excel workbook that holds the same data as the data matrix file, but also holds the probe-set annotation at the time we analyzed the data, and some simple statistical calculations, which select subsets of the probe-sets as differentially expressed. Consumers should consider obtaining more up-to-date probe-set annotation for the array platform. We also provide supplementary excel files that show our simple analysis of GSE6008, which consists of 99 human ovarian tumor samples of 4 types, and 4 normal ovary samples, where we fit an ANOVA model to the 5 groups. In yet another supplementary file we show the correlation between each human tumor and mouse tumor, where we correlate the difference in log2-transformed values of each tumor from the average of the normals for the same species, for just those genes that were 1-to-1 best homologs according to build 68 of NCBI's Homologene, in order to see how much the human tumors resemble the mouse tumors. Six oviductal tumor samples from 6 mice with conditional knockout of Apc and Pten driven by an Ovgp1-iCre-ERT2 transgene , 5 ovarian tumor samples from 4 mice with conditional knockout of Apc and Pten driven by injection of AdCre into both ovarian bursal cavities, and 4 normal ovary samples from 4 mice.

Project description:We treated the following 3 groups of 8-16 week old mice with tamoxifen: 1) CDX2P-CreERT2, Apc flox/+, Kras LSL-G12D/+, Trp53 flox/flox mice (n=6); 2) CDX2P-CreERT2, Apc flox/+, Kras LSL-G12D/+,Trp53 R270H/flox mice (n=6); 3) Wild-type control mice (n=3). The CDX2P-CreERT2 transgene expresses a tamoxifen (TAM)-regulated Cre protein (CreERT2) under control of human CDX2 regulatory sequences, allowing for TAM-inducible targeting of flox alleles in adult mouse terminal ileum, cecum, and colon epithelium. Treating the mice having CDX2P-CreERT2 transgene with tamoxifen permits the Cre recombinase to enter the cell nucleus and recombine the floxed alleles for Apc, Kras, and Trp53, resulting in deletion mutations in Apc and Trp53, and an activating, oncogenic mutation in Kras (G12D mutation). The Trp53 R270H allele carries a constitutive R270H mutation, which is the mouse equivalent of human TP53 R273H mutation. Laser capture microdissection was used to obtain adenocarcinoma tissue samples from the first two groups of mice and normal tissue from control mice. RNA was purified and targets for Affymetrix arrays were synthesized from the mRNAs. We used Affymetrix Mouse Gene 2.1 ST plate arrays, which hold 41345 probe-sets, but we largely analyzed just those 24562 probe-sets that were mapped to Entrez gene IDs. Raw data was processed with the Robust Multi-array Average algorithm (RMA). Data is log2-transformed transcript abundance estimates. We fit a one-way ANOVA model to the 3 groups of samples. We supply a supplementary excel workbook that holds the same data as the data matrix file for those 24562 probe-sets, but also holds the probe-set annotation at the time we analyzed the data, and some simple statistical calculations, which selects subsets of the probe-sets as differentially expressed between pairs of groups. It also shows data and analysis from a separate experiment of RNA purified from organoids cultured from samples from 3 groups of mice similar to those here, as well as an extra group of mice with CDX2P-CreERT2 Apc flox/flox genotype. It also joins a statistical summary of differences between 9 human tumors with TP53 missense mutations at codon 273 and 36 tumors with TP53 null mutations assayed with RNA-seq by the TCGA project. A separate supplementary file of the TCGA data is also provided. Consumers should consider obtaining more up-to-date probe-set annotation for the array platform.

Project description:For organoid preparation, we first treated the following 4 groups of 8-12 week old mice with tamoxifen: 1) CDX2P-CreERT2, Apc flox/+, Kras LSL-G12D/+, Trp53 flox/flox mice (n=2); 2) CDX2P-CreERT2, Apc flox/+, Kras LSL-G12D/+,Trp53 R270H/flox mice (n=3); 3) CDX2P-CreERT2, Apc flox/flox mice (n=3); 4) Wild-type control mice (n=4). The CDX2P-CreERT2 transgene expresses a tamoxifen (TAM)-regulated Cre protein (CreERT2) under control of human CDX2 regulatory sequences, allowing for TAM-inducible targeting of flox alleles in adult mouse terminal ileum, cecum, and colon epithelium. Treating the mice having CDX2P-CreERT2 transgene with tamoxifen permits the Cre recombinase to enter the cell nucleus and recombine the floxed alleles for Apc, Kras, and Trp53, resulting in deletion mutations in Apc and Trp53, and an activating, oncogenic mutation in Kras (G12D mutation). The Trp53 R270H allele carries a constitutive R270H mutation, which is the mouse equivalent of human TP53 R273H mutation. Colon tumors were induced by TAM treatment in all the mice from the first three groups and organoids were derived from the tumors of each mouse. We also derived organoids from the normal colon epithelium in the 4th group of mice as controls. All organoids were generated and propagated using a slightly modified TMDU protocol as described in PMID:20872391. Organoids were cultured for 4 days and then harvested. RNA was purified from the organoids, and targets for Affymetrix arrays were synthesized from the mRNAs. We used Affymetrix Mouse Gene 2.1 ST plate arrays, which hold 41345 probe-sets, but we largely analyzed just those 24562 probe-sets that were mapped to Entrez gene IDs. Raw data was processed with the Robust Multi-array Average algorithm (RMA). Data is log2-transformed transcript abundance estimates. We fit a one-way ANOVA model to the 4 groups of samples. We supply a supplementary excel workbook that holds the same data as the data matrix file for those 24562 probe-sets, but also holds the probe-set annotation at the time we analyzed the data, and some simple statistical calculations, which selects subsets of the probe-sets as differentially expressed between pairs of groups. It also shows data and analysis from a separate experiment of RNA purified directly from 3 groups of mice with genotypes like those of the organoid data except that no group of mice with CDX2P-CreERT2 Apc flox/flox genotype were used. It also joins a statistical summary of differences between 9 human tumors with TP53 missense mutations at codon 273 and 36 tumors with TP53 null mutations assayed with RNA-seq by the TCGA project. A separate supplementary file of the TCGA data is also provided. Consumers should consider obtaining more up-to-date probe-set annotation for the array platform.

Project description:We tamoxifen treated 8-12 week old mice that had floxed alleles of the following: 1) both Apc alleles (giving rise to Apc truncation/inactivation); 2) both Cdx2 alleles (giving rise to Cdx2 inactivation; 3) one Braf allele, that upon Cre-mediated recombination gives a Braf V600E mutant allele (details below), and 4) the combination of both the Cdx2 alleles and the BrafV600E allele. All four of those groups also had a CDX2P-CreERT2 transgene that expresses Cre recombinase fused to a tamoxifen-regulated fragment of the estrogen receptor ligand binding domain. CreERT2 expression occurs only in tissues where the Cdx2 gene is expressed, which is almost exclusively in adult mouse cecum and colon epithelium. A fifth group of mice had the floxed Cdx2 alleles, but no CDX2P-CreERT2 gene. Treating the mice having CDX2P-CreERT2 with tamoxifen permits the Cre recombinase to enter the cell nucleus and recombine the Apc, Braf, and/or Cdx2 alleles containing loxP sequence elements. Mice were treated with intraperitoneal injection of tamoxifen dissolved in corn oil. Three mice per group were used. The control mice did not develop tumors or any morphological or histological changes in their epithelium, but their colons were used to create the 3 control samples. To obtain the BrafV600E allele we used a genetically engineered mouse line previously described by Dankort et al. (Genes Dev 2007, 21:379-84) that can express the BrafV600E mutant protein following Cre-mediated recombination. The Braf(CA) (Braf-Cre-activated) allele mice carry a gene-targeted allele of Braf, where Braf sequences from exons 15-18 are present in the normal mouse Braf intron 14, followed by a mutated exon 15 (carrying the V600E mutation). The exon 15-18 sequence element is flanked by loxP sites. In the absence of Cre-mediated recombination, the Braf(CA) allele expresses a wild type Braf protein. Following Cre-mediated recombination, the Braf exon 15-18 element is removed, and the Braf(CA) allele then encodes the Braf V600E protein (from the introduced mutated exon 15). RNA was purified from tumor or normal tissue, and targets for Affymetrix arrays were synthesized from the mRNAs. We used Affymetrix Mouse Gene 2.1 ST arrays, which hold 41345 probe-sets, but we largely analyzed just those 25216 probe-sets that were mapped to Entrez gene IDs. Raw data was processed with the Robust Multi-array Average algorithm (RMA). Data is log2-transformed transcript abundance estimates. We fit a one-way ANOVA model to the five groups of samples. We supply a supplementary excel workbook that holds the same data as the data matrix file, but also holds the probe-set annotation at the time we analyzed the data, and some simple statistical calculations, which selects subsets of the probe-sets as differentially expressed between pairs of groups, as well as significant Cdx2-/- by Braf V600E interactions. It also gives the homologous human gene IDs we used for enrichment testing, which were 1-to-1 best homologs according to build 68 of NCBI's Homologene. A second supplementary sheet shows the data we enrichment tested after collapsing to distinct human homologs, joins of the results of tests with GSE4045 data and of tests with TCGA data to the mouse genes, and the intersections of selected genes in those data set with our gene selections in mouse. Consumers should consider obtaining more up-to-date probe-set annotation for the array platform.

Project description:Hypertension is a common cardiovascular disease that is related to genetic and environmental factors, but its mechanisms remain unclear. DNA methylation, a classical epigenetic modification, not only regulates gene expression but is also susceptible to environmental factors, linking environmental factors to genetic modification. Therefore, globally screening differential genomic DNA methylation in hypertensive patients is important for investigating novel hypertension mechanisms.Differential genomic DNA methylation in hypertensive, prehypertensive, and healthy control individuals was screened using the Illumina 450K BeadChip and verified by pyrosequencing. Plasma oviduct glycoprotein 1 (OVGP1) levels were determined using an enzyme-linked immunosorbent assay. Ovgp1 transgenic and knockout mice were generated to analyze the function of OVGP1. The blood pressure levels of the mouse models were measured using the tail-cuff system and radiotelemetry methods. The role of OVGP1 in vascular remodeling was determined by vascular relaxation studies. Protein–protein interactions were investigated using a pull-down/mass spectrometry assay and verified with coimmunoprecipitation and pull-down assays. We found a hypomethylated site at cg20823859 in the promoter region of OVGP1, and the plasma OVGP1 levels were significantly increased in hypertensive patients. This finding indicates that OVGP1 is associated with hypertension. In Ovgp1 transgenic mice, OVGP1 overexpression caused an increase in blood pressure, dysfunctional vasoconstriction and vasodilatation, remodeling of arterial walls, and increased vascular superoxide stress and inflammation, and these phenomena were exacerbated by angiotensin II infusion. In contrast, Ovgp1 deficiency attenuated angiotensin II-induced vascular oxidase stress, inflammation, and collagen deposition. These findings indicate that OVGP1 is a prohypertensive factor that directly promotes vascular remodeling. Pull-down and coimmunoprecipitation assays showed that myosin heavy chain II-A (MYH9) interacted with OVGP1, whereas inhibition of MYH9 attenuated OVGP1-induced hypertension and vascular remodeling. CONCLUSIONS: Hypomethylation at cg20823859 in the promoter region of OVGP1 is associated with hypertension and induces upregulation of OVGP1. The interaction between OVGP1 and MYH9 contributes to vascular remodeling and dysfunction. Therefore, OVGP1 is a prohypertensive factor that promotes vascular remodeling by binding with MYH9.

Dataset Information

Oviductal vs. ovarian epithelial transformation yields very different tumor phenotypes in Apc fl/fl;Pten fl/fl mice

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets